Astaxanthin is a compound that is conventionally used for reviving the colors of farm-raised fish such as salmon, trout, and sea bream, and recently considered about application to health food owing to its antioxidant activity. Astaxanthin is obtainable from natural products by extraction from crustaceans such as south pole euphausiid or culturing of yeast, microalgae and the like, however, nowadays chemically synthesized astaxanthin is widely used on account of stability of supply and cost problems. However, since chemically synthesized astaxanthin is susceptible to contamination of impurities from the process, particularly, of deleterious substances used in synthesis reaction, provision of natural products is demanded from the view point of safety. On the other hand, naturally occurring astaxanthin suffers from the problems in stability of supply and cost, and astaxanthin obtained by culturing of yeast or microalgae, in particular, faces a problem of contamination of fatty acid ester as a byproduct. Also it faces a problem that complicated process should be taken for extraction because of rigid cell walls.
For improving this, Japanese Patent Laid-Open Publication No. Hei 7-184668 (referred to as Patent Document 1) discloses a production method based on culturing of marine bacteria belonging to a genus Agrobacterium (for example, strain N-81106 (deposition number: FERM P-14023) later reclassified into bacteria belonging to Paracoccus, see Non-patent documents 1, 2 and 3). The invention is advantageous in that astaxanthin can be easily obtained only by mixing and stirring an organic solvent such as acetone and bacterial cells after harvesting cultured cells of marine bacteria. However, the astaxanthin obtained by culturing the above bacterium is as small as about 0.1 mg per 1 litter of culture solution, so that improvement in production amount is desired. Regarding a production process of astaxanthin based on culturing of bacteria, description can be found in Japanese Patent Laid-Open Publication No. 2001-352995 (referred to as Patent document 2) about a production method using soil bacteria, wherein 128 mg per 1 litter is produced. However, for use in aquafarming of marine products such as trout and sea bream, it would be preferable to use a microorganism obtained from the environment in which the marine product grows from the view point of safety. Therefore, improvement in production process by such marine microorganisms is desired.
In order to solve these problems, one conventional art reports conducting mutation breeding by using a marine bacterium strain N-81106, belonging to a genus Agrobacterium (deposition number: FERM P-14023) to acquire strain TSUG1C11 (deposition number: FERM P-19416) that produces 19.4 mg of astaxanthin per 1 litter of culture solution (see Patent document 3). It also reports acquisition of strain TSN18E7 (deposition number: FERM P-19746) that produces 200 mg of astaxanthin and 400 mg of total carotenoid per 1 litter of culture solution through further mutation breeding (see Patent document 3). From the viewpoint of industrial production, however, it is desired to produce more carotenoids such as astaxanthin, and hence strains having improved productivity are desired.
In industrial culturing of microorganisms, in particular, culturing of Escherichia coli or yeast, it is known that objective substances or microbial cells can be obtained at higher yield by a fed-batch culture method in which components of culture medium are supplemented during culture, than by a batch culture method in which necessary nutrients are loaded at once. Here, supplementing ingredients of culture medium is called “feeding”. With the feeding culture, nutrients to be supplemented can be desirably controlled, usually to lower concentrations. When production of objective substances or growth is prevented or when a byproduct such as alcohol and organic acid is produced by high concentration of substrates, such phenomenon can be suppressed by the control as described above. Particularly well known are suppression of production of objective substances, called “catabolite repression” observed when concentration of saccharides such as glucose in ingredients of culture medium is high, and suppression of growth of microorganism caused by toxicity of methanol and the like alcohols used at concentration. It is also known that when glucose is used at high concentration, ethanol and acetic acid accumulate for the case of yeast and Escherichia coli, respectively, and when they exceed 20 g/L and 5 g/L, respectively, growth is suppressed by byproducts. Production of byproducts is unfavorable because it not only suppresses growth but also deteriorates quality of objective substance and makes purification difficult. However, these findings are for phenomena observed mainly in culturing of Escherichia coli and yeast, and there is no finding about the effect of concentration of nutrient in carotenoid producing microorganisms which are objectives of the present invention. As a subject nutrient, saccharides and the like carbon sources which are greatly consumed can be exemplified. However, since the consumption rate varies depending on the growth condition of microorganism, in order to keep the concentration of carbon source constant during culturing, it is necessary to control the feeding amount while monitoring the growth condition of microorganism by an appropriate means. Various proposals have been made to achieve this. For example, a method of feeding carbon source is known which uses consumption of oxygen as an index. According to this method, consumption of oxygen is determined from a difference in oxygen level between inlet gas and outlet gas. However, since measurement of oxygen concentration contains relatively great error and has a drawback of slow response, microbial activity during culture cannot be estimated accurately. This makes it difficult to control when an unexpected change occurs. As a method based on an analysis of composition of outlet gas, a method in which feeding is executed while taking respiration quotient (RQ) as an index is also known. Respiration quotient is an index that shows a ratio of fermentation and respiration in culturing of yeast, for example, and advantageously reflects metabolism condition of microorganism (see, for example, Non-patent document 4). As to microorganisms other than yeast, however, the efficacy is not clear. Respiration quotient is calculated from differences in oxygen concentration and carbon dioxide gas concentration between inlet gas and outlet gas. Therefore, not only there arises a problem concerning oxygen concentration measurement as described above, but also there arises a need for calculation based on two index measurements of oxygen concentration and carbon dioxide concentration, making data processing relatively complicated.
Also known is a method which uses change in pH or change in dissolved oxygen (DO) as a physicochemical index, however, such method has a problem of low response speed of a sensor, and faces a problem that in the case of carbon source depletion, a stress due to the depletion occurs to cause change in biological metabolic activity because of the slow response to compensation for the depletion. A method using an on-line glucose analyzer is disadvantageous in long-term stable control because of influences of required sampling amount, analyzing time, accuracy, stability, liquidity and the like. A method using an on-line laser turbidity meter is disadvantageous in that accuracy is deteriorated when the density of bacterial cells is high (see, for example, Non-patent document 5).
In view of the above, not only proposal of a new method is demanded, but also there is a problem that efficacy of the above indexes on carotenoid producing bacteria which are subjects of the present invention is not known at all because the above methods are developed for yeast, Escherichia coli and the like.    [Patent document 1] Japanese Patent Laid-Open Publication No. Hei 7-184668    [Patent document 2] Japanese Patent Laid-Open Publication No. 2001-352995    [Patent document 3] Japanese Patent Laid-Open Publication No. 2005-58216    [Non-patent document 1] Internet (website of Marine Biotechnology Institute), Biotechnology Institute, MBIC (cell strain collection database), [online], date of published unknown, sections of “characteristics”, “strain name” and “16s” in page 9, [found by a search on Jun. 8, 2005], Internet    [Non-patent document 2] Internet (website of National Institute of Genetics, JAPAN) Research Organization of Information and Systems National Institute of Genetics, Japan DNA databank, “DNA Data Bank of Japan”, [online], Oct. 8, 2002, page 3, section of “ORIGIN”, [found by a search on Jun. 8, 2005], Internet    [Non-patent document 3] Internet (website of US National Institute of Health), National Institute of Health, National Center for Biotechnology Information, [online], Oct. 8, 2002, page 4, sections of “Source origin” and “Features, [found by a search on Oct. 18, 2005], Internet    [Non-patent document 4] Murayama and Takemoto, TOSOH Research report, No. 28, pp. 49-58, 1984.    [Non-patent document 5] Yamane, T et al., J. of Ferment. Bioeng., 75, 443, 1993